Abstract Diabetes is one of the major human diseases, now frequently referred to as diabetes epidemic. Chronic diabetes leads to serious complications such as diabetic nephropathy, which is characterized by decline in kidney filtration function and is the leading cause of end-stage renal disease. A widely accepted hypothesis is that in diabetes hyperglycemia accelerates the non- enzymatic chemical modification of proteins (glycation), perturbing protein function and causing diabetic complications. Proteins of extracellular matrix (ECM) are highly susceptible to glycation because of their very low turnover rates, which allow for the accumulation of modifications. The traditional view of protein glycation is that glucose directly reacts with lysine residues of a protein molecule forming an Amadori intermediate that undergoes oxidative conversion to different advanced glycation end products (AGE). In recent years, a new paradigm has emerged with the understanding that hyperglycemia causes increased plasma levels of reactive carbonyl species (RCS), most prominently, methylglyoxal (MGO) and 3-deoxyglucosone (3-DG), derived from oxidative degradation of glucose and glycated proteins. These compounds are much more reactive than glucose and can induce more rapid and more widespread pathogenic protein damage. This new paradigm is now supported by a number of clinical and epidemiological studies. However, the mechanisms underlying RCS generation and pathogenicity are largely unknown. We have made several novel findings which contribute to understanding of the mechanisms of this new paradigm and which set the direction of this renewal application. These include: a) discovery of new intermediates produced from glucose autoxidation and oxidation of Amadori intermediate, which question the classical mechanisms for production of MGO and 3-DG;and b) identification of a novel mechanism of perturbation of cell-ECM interactions triggered by MGO and 3-DG. Based on these findings we propose 1) to define novel pathways of MGO and 3-DG production;2) to define pathways of protein modification by MGO and 3-DG;to determine how 3) MGO and 3-DG or 4) MGO- and 3-DG- modified ECM can induce glomerular cell injury. The completion of these Aims will provide new insights into how RCS are generated in hyperglycemic milieu and how they may contribute to structural and functional damage of glomerular ECM and to cell-ECM interactions that underlie pathogenesis of diabetic nephropathy. Narrative Diabetes is one of the major human diseases. Hyperglycemia of diabetes causes increase in reactive carbonyl species (RCS), a phenomenon known as "carbonyl stress". We propose to investigate how RCS are generated in diabetes and how they cause protein and renal cell damage that underlie renal pathology.